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ORIGINAL ARTICLE
Novel approach to the microbial decontamination ofstrawberries: chlorophyllin-based photosensitizationZ. Luksiene and E. Paskeviciute
Institute of Applied Sciences, Vilnius University, Vilnius, Lithuania
Introduction
Fresh produce is becoming more popular all over the
world. Strawberries (Fragaria ananassa Duch.) are a pop-
ular and nutritious fruit worldwide. They have been
reported to contain the highest antioxidant capac-
ity among twelve fruits analysed (Wang et al. 1996;
Kahkonen et al. 2001). The main contributors to antioxi-
dant activity are phenolic compounds which have positive
effects for human health, preventing cancer, cardiovascu-
lar diseases, and age-induced oxidative stress (Olsson
et al. 2004). Anthocyanins are the most abundant flavo-
noids (phenolics) and are present at high level in mature
strawberries (Cordenunsi et al. 2005).
Short ripening and senescence periods of strawberries,
susceptibility to mechanical injury, contamination during
storage with fungi and bacteria reduces significantly their
shelf life. Strawberries spoilage losses can be as high as
40% (Satin 1996).
Furthermore, fresh produce has been increasingly
implicated as a vehicle for transmission of foodborne
illnesses. Such foodborne illnesses are estimated to result
in $ 6Æ9 billion of loss in productivity and medical
expenses to the US economy (ERS 2005). Several out-
breaks associated with consumption of strawberries have
been reported (FDA 1999). A variety of pathogenic bacte-
ria such as Listeria monocytogenes, Salmonella spp. as well
as pathogenic Escherichia coli strains may be present on
fresh fruits (Knudsen et al. 2001; Johannessen et al. 2002).
The most widely known postharvest treatments to
reduce microbial spoilage are low temperature and modi-
fied atmosphere packaging (Nielsen and Leufven 2008).
However, it has been reported that these technologies are
not effective enough and can have a negative impact on
Keywords
decontamination, nonthermal,
photosensitization, preservation, strawberry.
Correspondence
Zivile Luksiene, Institute of Applied Sciences,
Vilnius University, Sauletekio 10, 10223
Vilnius, Lithuania.
E-mail: [email protected]
2010 ⁄ 2179: received 1 December 2010,
revised 10 February 2011 and accepted 11
February 2011
doi:10.1111/j.1365-2672.2011.04986.x
Abstract
Aims: This study is focused on the possibility to control microbial contamina-
tion of strawberries by chlorophyllin (Na-Chl)-based photosensitization.
Moreover, photosensitization-induced effects on key quality attributes of trea-
ted strawberries was evaluated.
Methods and Results: Strawberries were inoculated with Listeria monocytogenes
ATCL3C 7644, soaked in 1 mmol l)1 Na-Chl for 5 min and illuminated for
30 min with visible light (k = 400 nm, energy density 12 mW cm)2). Results
indicated that the decontamination of strawberries using photosensitization
was 98% compared to control sample. Naturally occurring yeasts ⁄ microfungi
and mesophiles were inhibited by 86 and 97%, respectively. The shelf life of
treated strawberries was extended by 2 days. The total antioxidant activity of
treated strawberries increased by 19%. No impact on the amount of phenols,
anthocyanins or surface colour was detected.
Conclusions: Photosensitization may be an effective, nonthermal and environ-
mentally friendly microbial decontamination technique which expands the shelf
life of strawberries without any negative impact on antioxidant activity, and
phenols, anthocyanins or colour formation.
Significance and Impact of the Study: Experimental data support the idea that
Na-Chl-based photosensitization can be a useful tool for the future develop-
ment of nonthermal food preservation technology.
Journal of Applied Microbiology ISSN 1364-5072
1274 Journal of Applied Microbiology 110, 1274–1283 ª 2011 The Society for Applied Microbiology
ª 2011 The Authors
the quality of strawberries (Ayala-Zavala et al. 2007). For
instance, carbon dioxide treatment reduced anthocyanins
content and changed internal fruit colour (Gil et al.
1997). Washing alone or conventional sanitizers have
been shown to have limited efficacy at removing spoilage
and pathogenic bacteria from the surface (Yuk et al.
2006). There is a need to develop novel processing tech-
nologies that are more effective and do not diminish the
organoleptic properties and nutritional value of the
treated produce.
Photosensitization is a novel nonthermal and ecologi-
cally friendly treatment that involves the administration
of a photoactive compound (photosensitizer) and visible
light. After spraying of the photosensitizer on the surface
of fruit or vegetable, most pathogens and harmful bacte-
ria distributed on the surface of the fruit are able to bind
the photosensitizer. The following illumination of fruits
with visible light induces various photocytotoxic reactions
and death of surface-attached micro-organisms without
any harmful effects on environment (Luksiene 2005;
Luksiene and Zukauskas 2009). Na-Chl is a water-soluble
food additive (E141) and is used as food colourant, in
dietary supplements and in cosmetics (Lopez-Carballo
and Ocio 2008). According to our preliminary data,
Na-Chl interacts with bacterial wall, outer membrane and
after illumination destroys their integrity. This prevents
the opportunity to kill micro-organisms using a nonther-
mal technology.
This study is focused on the possibility to decontami-
nate strawberries by photosensitization from Gram-
Positive food pathogen L. monocytogenes, naturally dis-
tributed yeasts, microfungi and mesophiles. In addition,
photosensitization-induced effects on key quality attri-
butes of treated strawberries was evaluated.
Material and Methods
Pure culture of L. monocytogenes
Listeria monocytogenes ATCL3C 7644 was kindly provided
by the National Veterinary Laboratory (3rd passage of
ATCC7644-test organism) (Vilnius, Lithuania). The bac-
terial strain was cultured on Tryptone Soya Agar supple-
mented with 0Æ6% Yeast Extract (TSYEA) (Liofilchem,
Roseto degli Abruzzi, Italy) for 24 h at 30�C. For bacterial
suspension preparation, L. monocytogenes was grown
overnight (c. 14 h) at 37�C in 20 ml of Tryptone Soya
medium supplemented with 0Æ6% Yeast Extract (TSYE)
(Liofilchem), with agitation of 120 rev min)1 (Environ-
mental Shaker-Incubator ES–20; Biosan, Riga, Latvia).
The overnight bacterial culture was diluted 20 times by
the fresh medium (A = 0Æ164) and grown at 37�C to
mid-log phase (c. 1Æ16 · 109 colony forming units ml)1
(CFU ml)1), A = 0Æ9) in a shaker (120 rev min)1). Bacte-
rial optical density was determined in a 10Æ01 mm glass
cuvette at k = 540 nm (Hekios Gamma; ThermoSpec-
tronic, Cambridge, UK). Cells were then harvested by
centrifugation (20 min, 5000 g) (PC-6, Moscow, Russia)
and resuspended to c. 5Æ8 · 109 CFU ml)1 final concen-
tration in 0Æ1 mol l)1 phosphate buffer saline (PBS,
pH = 7Æ2). This stock suspension was accordingly PBS-
diluted to c. 1 · 107 CFU ml)1 and used for the further
photosensitization experiments.
Inoculation of L. monocytogenes on the surface
of strawberries
Strawberries (F. ananassa Dutch.) were purchased in a
local supermarket and used within 1 day. Prepared inocu-
lum (described above) of L. monocytogenes was poured
over strawberries and left for 30 min at room temperature
for cell attachment.
Photosensitization treatment
Naturally contaminated or pathogen-inoculated strawber-
ries were soaked in 1 mmol l)1 chlorophyllin Na-salt
(Na-Chl) (Roth, Karlsruhe, Germany) solution for 5 min.
The dried strawberries were placed in the treatment
chamber in a sterile Petri dish uncovered and exposed to
light intensity 12 mW cm)2 at k = 400 nm for 20 min.
The light source necessary for photosensitization was
constructed in the Institute of Applied Sciences of
Vilnius University. Light dose delivered to the surface of
sample was calculated as light intensity multiplied by
time (14Æ4 J cm)2). Light power density measurements
were carried out using a light energy 3 Sigma meter
(Coherent, Santa Clara, CA, USA) equipped with piro-
electrical detector J25LP04. Control samples were soaked
in Na-Chl solution but not illuminated in the chamber.
Illumination (400 nm) of unsoaked in Na-Chl samples
(other control) did not have any effect on survival of
micro-organisms.
Total aerobic micro-organisms count
Strawberry samples after treatment were separately mixed
with an appropriate volume of 0Æ1 mol l)1 PBS (1 g of
sample – 10 ml buffer) and homogenized in sterile
BagPage bags using a BagMixer (model MiniMix 100 VP,
Interscience, St. Nom, France). Total aerobic micro-
organism count was determined by serial dilutions (in
0Æ9% NaCl) plated on TSYEA and incubation at 30�C for
48 h. Total yeasts and microfungi count were determined
by serial dilutions (in 0Æ9% NaCl) plated on dichlo-
ran glycerol agar and incubation at 30�C for 72 h The
Z. Luksiene and E. Paskeviciute Decontamination of strawberries by photosensitization
ª 2011 The Authors
Journal of Applied Microbiology 110, 1274–1283 ª 2011 The Society for Applied Microbiology 1275
surviving cell populations were enumerated and expressed
by log10 (CFU g)1).
Evaluation of shelf life of strawberries
To evaluate shelf life of treated strawberries, one part of
strawberries was soaked in 1 mmol l)1 Na-Chl salt
solution, and the control in sterile distillated water.
Samples treated with Na-Chl salt were illuminated for
20 min, dried and stored in refrigerator (+6�C). The con-
trol samples were not illuminated and stored under the
identical conditions. Every experimental group consisted
of 30 berries (weight 10–15 g). Disease-free (shelf life) per-
iod of strawberries was evaluated visually by assessment of
colour change (rots, spots), induced by growth of spoilage
micro-organisms during 9 days after treatment with ber-
ries stored in a refrigerator (+6�C). The level of infected
berries was scored on a 1–6 scale. Results were expressed
as a disease index between 0 and 100 (0, no infection; 100,
all fruits are infected) (Kittemann et al. 2008).
Temperature measurement on the surface of berry
Precision Celsius temperature sensors (Deltha Ohm,
Padua, Italy) were used for temperature measurements on
the surface of strawberry.
Measurements of total antioxidant capacity
Total antioxidant capacity of strawberries was measured
by ferric reducing ability of plasma (FRAP) method
(Benzie and Strain 1996). Extracts for measurement were
prepared by homogenization of 1 g of fruit with 50 ml
96% alcohol (Minimix, Interscience). FRAP working solu-
tion included 0Æ3 mol l)1 acetate buffer (pH 3Æ6), 0Æ01
mol l)1 2, 4, 6-tripyridyl-s-triazine (TPTZ) in 0Æ04 mol l)1
HCl and 0Æ02 mol l)1 FeCl3Æ6H2O in distilled water. For
measurement of antioxidant activity, 1Æ5 ml of FRAP
reagent and 50 ll of sample solution were mixed. The
reading was performed every 30 s up to 5 min at 593 nm
(Hekios Gamma; ThermoSpectronic), 1-cm light path. Fe
(II) standard solution was tested in parallel.
Total soluble phenols assay
Samples of strawberries (3–10 g) were homogenized for
1 min at maximum speed in a Minimix with 30–100 ml
of mixture containing acetone, distilled water and acetic
acid (70 : 29Æ5 : 0Æ5). Samples were mixed and allowed to
stand for 1 h at room temperature. Extracts were centri-
fuged at 1640 g for 15 min (Micro 200; Hettich, Beverly,
MA, USA), and supernatant was used for total phenols
(TP) assay.
TP concentration was measured using the Folin-
Ciocalteu assay (Asami et al. 2003). In brief, 5 ml of
distilled water, 0Æ5 ml of sample and 1 ml of Folin-
Ciocalteu reagent were mixed and left at room tempera-
ture for 5 min. Then, 10 ml of 7% sodium carbonate
solution was added and solution was filled to 25 ml final
volume by the addition of distilled water. Solution was
mixed well and left at room temperature for 2 h. Then,
the mixture was filtered through 8-layer cheesecloth. After
that, the TP concentration using a spectrophotometer
monitoring 750 nm (Hekios Gamma; ThermoSpectronic)
was measured. TP content was standardized against gallic
acid and expressed as milligrams per litre of gallic acid
equivalents.
Total anthocyanins assay
Samples weighing 10 g of treated strawberries were
blended in a food processor for 1 min with 75 ml of a
mixture of methanol, acetic acid and distilled water at a
ratio of 25 : 1 : 24. Mixture was centrifuged at 2000 g for
20 min (Micro 200). The supernatant was removed and
mixed with 75 ml M : A : W then centrifuged again, and
the supernatant was separated. Each sample was extracted
3 times. Optical density was measured using 1 cm path
length quartz cuvette at 535 nm (Hekios Gamma;
ThermoSpectronic) (Tiwari et al. 2009).
Measurement of colour
Possible changes of strawberry colour after photosensitiza-
tion were evaluated from absorption spectrum measuring
optical density (OD) of the sample in visible region of
spectrum. Samples weighing 10 g of fresh berry were
blended in a food processor for 1 min with 75 ml of a
mixture of methanol, acetic acid and distilled water
(M : A : W) at a ratio of 25 : 1 : 24. The mixture was
then centrifuged at 2000 g for 20 min (Micro 200). Opti-
cal density (310–650 nm) was measured using 1 cm path
length quartz cuvette with spectrophotometer (Hekios
Gamma; ThermoSpectronic). Each sample was extracted 3
times.
Statistics
The experiments were carried out in triplicate for each set
of exposure, using different batches of strawberries. The
data were analysed with Origin 7.5 software (OriginLab
Corporation, Northampton, MA, USA). A standard error
was estimated for every experimental point and marked
in a figure as an error bar. Sometimes the bars were too
small to be visible. To estimate the significance of inacti-
vation of L. monocytogenes on the surface of strawberries
Decontamination of strawberries by photosensitization Z. Luksiene and E. Paskeviciute
1276 Journal of Applied Microbiology 110, 1274–1283 ª 2011 The Society for Applied Microbiology
ª 2011 The Authors
(Fig. 1) and to compare the amounts of soluble phenolics
and anthocyanins in strawberries (Fig. 4b,c), analysis of
variance (anova) with Bonferroni test was used. To esti-
mate the significance of inactivation of total mesophiles,
yeasts and fungi (Fig. 2a,b) on the surface of strawberries
by photosensitization, shelf life of strawberries (Fig. 3)
and the effect of photosensitization on the amount of
total antioxidants (Fig. 5a) Student’s t-test was used.
Results
Photosensitization-based inactivation of L. monocytogenes
inoculated on the surface of strawberry
The data depicted in Fig. 1 indicated that the population
of inoculated Listeria in untreated control strawberry
group grew to 6Æ8 log. Soaking of berries in Na-Chl solu-
tion or just illumination with light was indifferent and
did not affect the bacterial survival. As little as
1 mmol l)1 Na-Chl-based photosensitization reduced the
population of Listeria by 1Æ8 log (98%).
Photosensitization-based inactivation of total aerobic
mesophils, yeasts and fungi
Subsequently, it was determined whether naturally sur-
face-distributed mesophiles, microfungi and yeasts were
susceptible to photosensitization. Data presented in
Fig. 2a indicated that the growth of total aerobic meso-
philes in control group increased from 4 log to 8 log
during 8 days. In treated strawberries, the amount of
mesophiles reduced by 1Æ7 log (97%) and increased less
(6 log) during 8 days in comparison with control (8 log).
No significant changes of growth rate (l) of mesophiles
were observed during 8 days after treatment: in control
l = 1Æ15 compare it in treated samples l = 1Æ10.
Data presented in Fig. 2b indicate that 1 mmol l)1
Na-Chl-based photosensitization can reduce the amount
of yeasts and fungi in the strawberries by 0Æ86 log (86%).
Examination of regrowth of micro-organisms during
8 days after treatment indicates that in control group the
yeasts and microfungi increase from 2Æ1 log to 7Æ1 log
during 8 days, where as in treated group their amount
during the same period reached 5Æ8 log. The regrowth
rate of yeast and fungal survivors slightly decreased (in
control l = 1Æ44, in treated samples l = 1Æ27).
7
6
5
4
3
2
1
0
Cel
l Num
ber,
Log
10
Figure 1 Inactivation of Listeria monocytogenes ATCL3C 7644 on the
surface of strawberries by photosensitization with 1 mmol l)1 Na-Chl
(incubation time – 5 min, illumination time – 20 min). ( ), control; ( ),
Na-Chl without light and ( ), after photosensitization with Na-Chl.
9
8
7
6
5
4
3
2
1
00 2 4 6 8
Time (days)
Cel
l num
ber
(Log
10 C
FU
g–1
)
8
7
6
5
4
3
2
1
00 2 4 6 8
Time (days)
Sur
viva
l fra
ctio
n (L
og10
CF
U g
–1)
(a)
(b)
Figure 2 Inactivation and regrowth of total mesophils (a), yeasts and
fungi (b) on the surface of strawberries by photosensitization with
1 mmol l)1 Na-Chl during 8 days after treatment (incubation time –
5 min, illumination time – 20 min, storage time 8 days at +6�C). ( )
control and ( ) after photosensitization with Na-Chl.
Z. Luksiene and E. Paskeviciute Decontamination of strawberries by photosensitization
ª 2011 The Authors
Journal of Applied Microbiology 110, 1274–1283 ª 2011 The Society for Applied Microbiology 1277
Evaluation of shelf life of strawberries after
photosensitization
It is obvious that the most important advantage of any
antimicrobial technology is ability to extend the self-life
of treated berries. Shelf life of berries was evaluated visu-
ally from surface colour changes. Thus, as depicted in
Fig. 3, the disease-free period of treated strawberries was
prolonged about 2 days in comparison with control. This
is a significant effect as the Na-Chl concentration was not
high (1 mmol l)1). Furthermore, some delay of disease
induction in the treated strawberries was observed.
Measuring the extension of shelf life of treated straw-
berries by Kittemann et al. (2008) system (1–6 scale),
5-day storage makes all control berries (100%) infected
and disease index (N) was the highest N = 6, whereas just
25% of treated berries were damaged by spoilage micro-
organisms, thus disease index was significantly lowered
(N = 1Æ5).
Measurement of strawberry surface temperature
The temperature kinetics on the surface of berry during
treatment was evaluated using specially attached surface
digital attacher inside the treatment chamber. Data pre-
sented in Fig. 4 clearly indicate that during 20 min of
treatment the temperature on the surface of strawberry
increases very slowly and does not exceed 27�C.
Total antioxidant activity
According to the obtained results depicted in Fig. 5a, the
total antioxidant activity in control berries was 18 mmol
Fe2+ ⁄ kg whereas in berries treated by photosensitization
(1 mmol l)1 Na-Chl) it increased to more than 22 mmol
Fe2+ ⁄ kg. This statistically significant increase (19%) in
total antioxidant activity was observed in the strawberries
immediately after photosensitization.
Evaluation of the amount of total phenolics and
anthocyanins
To estimate specific changes of nutritional quality of
strawberries, the amount of total phenolics and anthocya-
nins in the treated and control strawberries was evaluated.
As depicted in Fig. 5b, the amount of total soluble phen-
olics immediately after photosensitization (1 mmol l)1
Na-Chl) did not differ from control (1750 mg per 100 g
f.f.). Storage without treatment at +6�C in the refrigerator
reduced their amount to 1400 mg per 100 g f.f. In addi-
tion, the level of total anthocyanins in strawberries after
photosensitization treatment was the same as in control
berries (Fig. 5c). Storage without treatment of strawber-
ries in refrigerator for 24 h reduced the anthocyanin level
from 145 mg PGN ⁄ 100 g f.f. to 122 mg PGN ⁄ 100 g f.f. in
control as well as in treated strawberries.
Measurements of strawberry colour
The other important characteristic that can be influenced
by photosensitization is the appearance of berry, espe-
cially its colour. Thus, strawberries from the most effec-
tive treatments were analysed immediately after treatment
to determine whether photosensitization had any negative
effects on the colour of the strawberry. For this purpose,
100
120
80
60
40
20
00 1 2 3 4 5 6 7 8 9
Time (days)
Sur
viva
l dis
trib
utio
n (%
)
Figure 3 Shelf life of strawberries after photosensitization with
1 mmol l)1 Na-Chl in comparison with control. (–––) control and (.....)
after photosensitization with Na-Chl.
30
25
20
15
10
5
00 2 4 6 8 10 12 14 16 18 20 22
Time (min)
Tem
pera
ture
(°C
)
Figure 4 The increase in temperature on the surface of strawberries
placed in LED-based light prototype during 20 min of illumination.
Thermometer (Delta Ohm, Italy) was used for temperature measure-
ments on the surface of berry.
Decontamination of strawberries by photosensitization Z. Luksiene and E. Paskeviciute
1278 Journal of Applied Microbiology 110, 1274–1283 ª 2011 The Society for Applied Microbiology
ª 2011 The Authors
absorption spectroscopy was used to analyse the spectrum
of berry extract in visible region. It is evident from Fig. 6
that no significant colour changes are detected over all
visible spectrum region, meaning that photosensitization
has no impact on strawberry colour.
Discussion
During the past decade, the emphasis in postharvest fruit
protection has shifted from using chemicals to various
alternative techniques including biological control
(Sharma et al. 2009) and physical methods such as con-
trolled atmosphere (Zheng et al. 2008; Sandhya 2010) or
irradiation (Dionisio et al. 2009).
To compare, irradiation of strawberries with 2–3 kGy
irradiation was found to suppress fungi on stored berries
and more than double their shelf life, but unfortunately
gave rise to changes in strawberry texture, cell wall com-
position and colour (Yu et al. 1995). Actually, ionizing
radiation has been approved for decontamination of food
in the USA. However, it is strongly discouraged in EU as
consumers mostly prefer nonirradiated products (Neyssen
2000).
Marquenie et al. (2003) studied the combined effect of
three physical methods – high-power pulsed light, heat
and UV-C illumination on strawberry decontamination
from fungus Botrytis cinerea. Their results revealed that
pulsed light alone was ineffective against selected fungus,
although combined treatment of all three techniques
reduced visually B. cinerea mycelia and did not affect fruit
firmness. This technique also prolonged disease-free
period increasing the shelf life by 1–2 days.
4·4
4·0
3·6
3·2
2·8
2·4
2·0
1·6
1·2
0·8
0·4
0·0300 350 400 450 500 550 600 650
Wavelength (nm)
Opt
ical
den
sity
Figure 6 Strawberry colour changes after Na-Chl photosensitization
treatment: absorption spectrum of strawberry extract samples in
visible region. ( ) control; ( ) Na-Chl without light and ( ) after
photosensitization with Na-Chl.
24
22
20
1816
14
12
10
8
6
4
2
0
mm
ol F
e2+ k
g–1 fr
uit
2000
1800
1600
1400
1200
1000
800
600
400
200
0Control
Tot
al s
olub
le p
heno
lics
(mg
per
100g
f.f)
Na-chlwithout light
Na-chl+5 min light
160
140
120
100
80
60
40
20
0Control Na-chl salt
without lightNa-chl salt
with 5 min light
Tot
al a
ntho
cyan
ins
(mg
PG
N p
er 1
00g
f.f)
(a)
(b)
(c)
Figure 5 The amount of total antioxidants (a), soluble phenolics (b)
and anthocyanins (c) in strawberries after 1 mmol l)1 Na-Chl-based
photosensitization in comparison with control during 0–24 h after
treatment keeping them at +6�C. ( ) control; ( ) after photosensitiza-
tion with Na-Chl; ( ) 0 h; ( ) 12 h and ( ) 24 h.
Z. Luksiene and E. Paskeviciute Decontamination of strawberries by photosensitization
ª 2011 The Authors
Journal of Applied Microbiology 110, 1274–1283 ª 2011 The Society for Applied Microbiology 1279
Allende et al. (2007) determined the effect of UV-
C light, gaseous O3, super atmospheric O2 and
CO2-enriched atmospheres applied individually and in
combination on the shelf life of strawberries. Individual
treatments did not affect the microbial contamination,
whereas a combination reduced the growth of yeast and
microfungi by 1 log for up to 5 days and prolonged the
storage period, respectively.
Data obtained in our previous studies revealed that
photosensitization-based treatment inactivated (6 log)
food pathogens L. monocytogenes ATCL3C 7644 and Bacil-
lus cereus ATCC 12826 in vitro when aminolevulinic acid
(Buchovec et al. 2009; Le Marc et al. 2009; Luksiene et al.
2009; Buchovec et al. 2010) or Na-Chl (Luksiene et al.
2010a,b) was used as photosensitizers. Moreover, such
microbial conditions as spores and biofilms were suscepti-
ble to this treatment (Luksiene et al. 2010a,b). Effective
photosensitization-induced inactivation of microfungi and
yeasts in vitro has been reported in our previous studies
(Luksiene 2005; Luksiene et al. 2005). These results
prompted us to investigate further the susceptibility of
pathogens, yeasts, microfungi and mesophiles to this
treatment in real food systems.
According to the data obtained, photosensitization-
based treatment can decontaminate strawberry from the
inoculated Listeria by 98% (Fig. 1). Mesophiles naturally
distributed on the surface of strawberries have been inac-
tivated by 97% (Fig. 2a). It is important to note that
spoilage yeasts and microfungi showed slightly lower sus-
ceptibility to photosensitization and were inactivated by
86% (Fig. 2b). These data are in line with results of other
studies (Anderson et al. 2000). The overall reduction in
this microbial contamination prolongs the disease-free
period of berries by 2 days (Fig. 3).
The irregularity and the different light-reflecting prop-
erties of the illuminated strawberry surfaces can possibly
account for the different antimicrobial efficiency in vitro
and treating food matrix (Paskeviciute et al. 2009).
Taking into account that photosensitization efficiency
depends on illumination time and light intensity, the
obtained antibacterial efficiency can be significantly
enhanced by usage of more powerful light sources (light
emitting diodes-LED) or increase in illumination time
(Luksiene and Buchovec 2009).
Distinguishing feature of photosensitization is its non-
thermal action. It should be noted that there was no
significant temperature increase on the surface of straw-
berries which would be dangerous to the quality of fruit
after photosensitization treatment (Fig. 4). During 20 min
of treatment, the temperature on the surface of strawber-
ries changed from 20 to 25�C. It must be emphasized that
to find a physical antimicrobial technique without
thermal effects is a complicated task. For instance,
high-power pulsed light is an effective disinfection
method, but temperature increase will occur. The temper-
ature on the surface of strawberry increased to 80�C after
60 s of high-power pulsed light treatment (Bialka and
Demirci 2008).
The main beneficial properties of fruits and vegetables
have been partially attributed to the presence of antioxi-
dant compounds (Tulipani et al. 2008). Antioxidants can
scavenge free radicals and reactive oxygen species which
usually induce toxic processes in the living cell including
oxidative damage to proteins and DNA, membrane lipid
oxidation, enzyme inactivation and gene mutation that
may finally lead to cancer genesis or other oxidative
cardiovascular or inflammatory diseases (Eberhardt et al.
2000). Therefore, it might be possible that photosensitiza-
tion being an effective antimicrobial treatment modality
can affect and result in some negative impact on the
strawberry nutritional properties. Thus, it was necessary
to investigate whether some changes of antioxidant activ-
ity took place after photosensitization in strawberries.
According to our data presented in Fig. 5a, photosensiti-
zation induced significant increase in total antioxidant
activity (19%) in strawberries, which could be associated
with enhanced cellular capacity to detoxify reactive oxy-
gen species. However, the mechanisms of these effects
have not been thoroughly elucidated so far. In fact, plant
cells usually keep the reactive oxygen species (ROS) level
under tight control by production or activation of scav-
enging enzymes (Bailly 2004).
Recently, such phenolic compounds as flavonoids
(anthocyanins), flavanols (catechins) and flavonols (quer-
cetin) have attracted increasing attention as potent an-
tioxidants. They can stimulate carcinogen-detoxifying
enzymes and counteract inflammatory processes (Parr
and Bolwell 2000). Because phenolic compounds, as effec-
tive antimicrobials and UV screens, are accumulated in
epidermal tissue, the question arise, does photosensitiza-
tion affect their content in the strawberry fruit? According
to our results, the photosensitization did not affect the
level of soluble phenolics, and storage for only 24 h at
+6�C diminished their amount from 1750 mg per 100 g
f.f. to 1400 mg per 100 g f.f. That is in agreement with
the results of (Nunes et al. 1995).
As anthocyaninss prevent cardiovascular and other dis-
orders (Zafra-Stone et al. 2007), to save their concentra-
tion stable in strawberries is important. Many factors
such as pH, light, oxygen, enzymes and high temperature
can induce anthocyanin degradation (Wang and Xu
2007). For instance, Tiwari et al. (2009) studied the effect
of ozone on strawberry juice anthocyanins and found that
ozone induced significant reductions in their content
(98Æ2%). Data obtained in this work clearly indicate that
the amount of anthocyanins after photosensitization did
Decontamination of strawberries by photosensitization Z. Luksiene and E. Paskeviciute
1280 Journal of Applied Microbiology 110, 1274–1283 ª 2011 The Society for Applied Microbiology
ª 2011 The Authors
not change after soaking of berries in Na-Chl or after
photosensitization and remained very close to the initial
content (140 lg g)1). Slight decrease was observed even
after storage 24 h at +6�C (121 lg g)1). Photosensitiza-
tion increased the total antioxidant activity in strawber-
ries, but this effect had no correlation with amount of
total phenolics or their constituent anthocyanins.
Gil et al. (1997) studied the effect of carbon dioxide
treatment on the amount of anthocyanins and other
polyphenols in strawberries. Their results revealed that
anthocyanins content of CO2-treated fruit was reduced as
compared with air-stored fruit. Odriozola-Serrano et al.
(2008) also found that high-intensity pulsed electric field
(HIPEF) affected strawberry nutritional qualities. The loss
of phenolic content over the storage time in HIPEF and
thermally processed strawberry juices was in the range of
21Æ5–24Æ1 mg per 100 ml after 56 days at 4�C.
Colour is one of the important quality indicators in
fresh strawberry appearance and greatly contributes to
fruit quality. The bright red colour of strawberry fruit is
because of the presence of anthocyanin pigments in the
fruit epidermis and cortex (Nunes et al. 1995). In addi-
tion, factors such as copigmentation, pH and anthocyanin
metabolism may play a significant role in the expression
of colour in strawberries (Gil et al. 1997). The most effec-
tive copigments flavonols are located in external tissue
(Mazza and Miniati 1993). To determine whether photo-
sensitization had any effect on the colour of strawberry,
the fruits were analysed immediately after treatment.
According to the obtained data (Fig. 6), the colour of
strawberry surface was not markedly affected by the pho-
tosensitization as no significant difference was detected
between absorption spectrum of treated and control
fruits. Other studies performed on decontamination of
strawberries using carbon dioxide treatment indicated
that fruit surface colour did not change, but remarkable
changes were observed in internal flesh colour (Gil et al.
1997). However, high-power pulsed light used to decon-
taminate strawberries did not induce changes in fruit skin
colour (Bialka and Demirci 2008) as light-based technolo-
gies including photosensitization act superficially.
Preliminary data were obtained on the taste of treated
berries. Twenty-one volunteers tested the taste of treated
strawberries and compared them with control ones.
Eighteen of 21 volunteers found no sweetness or firmness
changes in control and treated berries. It can be easily
explained as visible light alone is not producing any
effects, and Na-Chl is a well-known food additive.
Conclusions
Data obtained in this study clearly indicate that photosen-
sitization might be useful tool to decontaminate strawber-
ries from Gram-positive food pathogen Listeria, yeasts,
microfungi and mesophiles distributed on the surface of
strawberries. These reductions are comparable to, if not
greater than, the reductions obtained by other methods.
One of the main advantages of photosensitization is the
absence of any harmful effects on strawberry antioxidant
activity, total phenolics, flavanoids (anthocyanins) or
colour with significant extension of their shelf life
(2 days). In addition, no negative impact on the taste of
treated strawberries was found. Importantly, treatment is
nonthermal and environmentally friendly. Thus, photo-
sensitization has potential as an antimicrobial tool for the
treatment of, for example, ready-to-eat fruits, frozen
berries, pastry products or similar. More detail studies
need to be conducted on the quality and organoleptic
characteristics of the treated fruits in the future.
Acknowledgements
This study was financially supported by the European
Commission (FP6 STREP project HighQ RTE, No 023140).
The authors are thankful Dr V. Gudelis and I. Buchovec
for their contribution to this study.
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